Cytochrome P450's (CYPs) are a major source of metabolic drug interactions. The allosteric behavior of CYPs, including the major drug metabolizing isoform CYP3A4, continues to confound in vitro - In vivo correlations and the prediction of drug interactions based on in vitro data. The molecular mechanisms that confer allosteric behavior are incompletely understood, and improved mechanistic models are likely to improve predictive drug metabolism. Moreover, although allosteric behavior in vitro is well known and widely observed, examples of in vivo allosterism are limited. Among the multiple mechanisms that contribute, occupancy by small molecule drugs at a peripheral effector site of CYP3A4, and conformational changes induced by the electron transfer partner Cytochrome bs (Cyt bs), are likely to converge on a set of common effects, wherein the active site is more efficiently desolvated or 'well-packed' through protein-protein interactions or multiple drug binding. In turn this, hypothetically, leads to more efficient coupling of NADPH consumption and O2 reduction, concomitant with drug oxidation, with decreased 'uncoupling' to form reduced oxygen species. This proposal aims to: 1) increase our understanding of allosteric CYP mechanisms with model probe drugs, and 2) to complete a comparative, and mechanistic, analysis of CYP-Cyt b5 interactions. The fluorescent probes Nile Red and TNS provide a comparison of the active site hydration and steric constraints of CYP3A4 when the peripheral allosteric site is occupied or empty. Mass spectrometry and computational models provide methods to map binding interactions in Cyt b5-CYP complexes and the resulting conformational changes linked to effector functions of Cyt bs. Each of these approaches will be combined with functional studies to characterize reaction intermediates that determine the efficiency of the flux through the catalytic cycle. A major goal of these studies is to develop their translational utility in conceptually analogous studies with P-glycoprotein in Project 4 of this Program. In addition, the importance of in vivo allosteric effects remains speculative, and the final aim, aim 3, of this proposal will utilize a novel in vivo, human, clinical study to explore heterotropic effects between the antiepileptic drug carbamazapine and the antianxiety drug midazolam. This study not only seeks to establish definitive proof-of-principle for in vivo allosteric effects in human CYPs, but also to provide a platform experimental design to be exploited in other in vivo studies.